A highly effective vaccine that targets the asymptomatic sporozoite (SPZ) and liver stages of the most lethal human malaria parasite, Plasmodium falciparum (Pf), would be an ideal tool to prevent malaria infection, disease and transmission. After 3 decades of effort, the most effective subunit malaria vaccine only provides 50% protection against infection at 2 wks and 22% at 5 months after the last dose. PfSPZ are the only immunogens that induce sustained (at least 10-28 months), high level (>90%) protection against Pf. For decades the focus has been on irradiated (irr) PfSPZ. Recently, however, genetically attenuated SPZ disrupted in genes required for early/mid liver stage development have shown protection in mice. Furthermore, volunteers immunized by the bite of mosquitoes carrying fully infectious PfSPZ, and administered chloroquine to eliminate blood stage parasites, acquire protective immunity against liver stage parasites after exposure to 20 times fewer PfSPZ-infected mosquitoes than are required with mosquitoes carrying irrPfSPZ. This increased efficiency is likely due to increased abundance and diversity of parasite epitopes presented to the immune system during the mid/late liver stages. Clinical trials are underway or planned for PfSPZ-based vaccines, including PfSPZ administered with chloroquine. However, it would be ideal if the need for an antimalarial drug were eliminated. Furthermore, in a rodent malaria model the drug azithromcyin, which kills parasites at the late liver stage by disrupting apicoplast function, is more efficient than chloroquine in inducing protective immunity. We propose to eliminate the need for an antimalarial drug and improve the efficiency of induction of protective immunity by using a genetically attenuated strain of Pf lacking genes that are essential for development of only late liver and asexual blood stages. Using a newly developed, highly efficient method for genome editing based on customized zinc-finger nucleases, we will delete the Pf dxr and lspD genes that encode apicoplast enzymes deoxyxylulose 5-phosphate reductoisomerase (DXR) and methylerythritol phosphate cytidyltransferase (IspD), respectively. DXR catalyzes the 1st step in isoprenoid biosynthesis, followed by IspD, resulting in production of the essential metabolite isopentenyl diphosphate (IPP). DXR is essential for Pf blood stage growth. Inhibition of apicoplast development and isoprenoid biosynthesis can be reversed by supplementation with IPP allowing for generation of double knockout parasites. Knockout clones will be selected that generate acceptable numbers of gametocytes and PfSPZ when compared to wild type parasites, and Sanaria will produce purified, cryopreserved Pf?dxr+?lspD SPZ. Studies in hepatocytes and a new liver tissue model will enable our testing of the hypothesis that Pf?dxr+?lspD parasites have a profound, IPP-dependent, developmental arrest late during liver stage development when the apicoplast is most active. These studies will establish a vaccine candidate with optimal immunogenicity and a critical safety feature of being unable to sustain replication in erythrocytes if any parasites break through from the liver.